DNA Polymerases

ABSTRACT

A DNA polymerase mutant comprising a Taq DNA polymerase amino acid sequence with a mutation at one or more of the following selected amino acid positions: E189K, E230K, E507K, H28R, L30R, G38R, F73V, H75R, E76A, E76G, E76K, E90K, K206R, E315K, A348V, L351F, A439T, D452N, G504S, E507A, D551N, L552R, I553V, D578N, H676R, Q680R, D732G, E734G, E734K, F749V; wherein the polymerase mutant exhibits relative to wild-type DNA polymerase increased polymerase speed, increased affinity to DNA substrate and/or increased resistance to a DNA polymerase inhibitor; and wherein, when the mutation is E507K in combination with two or more further mutations or the mutation is Q680R in combination with four or more further mutations, at least one of the further mutations is at one of the selected amino acid positions; and when the mutation is I553V, this is not in combination with D551S.

FIELD OF THE INVENTION

The present invention relates to DNA polymerases, which possessincreased resistance to PCR inhibitors, increased affinity to DNAsubstrate and increased DNA polymerization efficiency.

BACKGROUND OF THE INVENTION

Polymerase chain reaction (PCR) is probably the most popular applicationin contemporary molecular biology and diagnostics. The key components ofPCR are thermostable DNA polymerases, which synthesize new DNAcomplementary to a DNA matrix. There are many different polymerases,which are used in PCR. Even though Taq DNA polymerase was the firstenzyme employed in PCR and many new enzymes were discovered since thattime, this polymerase continues to be the most popular and widely usedin majority of PCR applications due to its robustness and efficiency aswell as easy and cost efficient production process. Taq DNA polymerasehas been studied very intensively and there is a lot of biochemical aswell as structural data available on it. Within the course of thesestudies many different mutations of Taq DNA polymerase have been createdand studied, which in one or another way improve properties of thisenzyme. Some mutations are important for enzyme fidelity (U.S. Pat. No.6,395,524, U.S. Pat. No. 6,602,695 and U.S. Pat. No. 5,614,365), somealter 5′-3′ exonuclease activity (U.S. Pat. No. 5,466,591), changeenzyme properties related to labeled nucleotide incorporation (Brandiset al., 1998), make the enzyme “cold sensitive” (Barnes and Kermekchiev,2000) or increase polymerase resistance to different PCR inhibitors(Kermekchiev and Barnes, 2004; Kermekchiev and Kirilova, 2006). Suchmutants of Taq DNA polymerase are useful in qPCR, DNA sequencing,amplification of DNA samples containing various PCR inhibitors (dye,blood, soil). For example, SYBR Green I intercalating dye is used inqPCR. This inhibits Taq DNA polymerase and can decrease PCR efficiencyand sensitivity. Increased polymerase resistance to SYBR Green I may beassociated with increased enzyme resistance to other PCR inhibitors fromblood and soil (Kermekchiev et al, 2009; Zhang et al, 2010).

Mutation at various different amino acid positions in the Taq DNApolymerase are known to improve various different properties. Theseinclude K219, K225, E520, D578, A608 (Brandis et al., 1998; Holliger etal., 2001), S515 (Hardin et al., 2006), A521, V529, Q592 (Brandis etal., 1998) and S543 (Jestin et al., 2005; Vatta et al., 2005). In oneexample, the positively charged Taq DNA polymerase mutation E507K isknown to improve the RNA target dependent activity by 50% compared tothe parent enzyme.

Currently PCR represents one of the fastest growing segments ofmolecular biology applications market. New applications for PCR and newvariants of PCR are being developed and introduced for research anddiagnostic applications, such as fast qPCR, digital PCR and directsample-to-PCR which require novel enzymatic properties. Therefore, thereis a need in the industry for Taq DNA polymerase derivatives possessingnovel, improved properties.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a DNA polymerasemutant comprising a Taq DNA polymerase amino acid sequence with amutation at one or more of the following selected amino acid positions:E189K, E230K, E507K, H28R, L30R, G38R, F73V, H75R, E76A, E76G, E76K,E90K, K206R, E315K, A348V, L351F, A439T, D452N, G504S, E507A, D551N,L552R, I553V, D578N, H676R, Q680R, D732G, E734G, E734K, F749V; whereinthe polymerase mutant exhibits relative to wild-type DNA polymeraseincreased polymerase speed, increased affinity to DNA substrate and/orincreased resistance to a DNA polymerase inhibitor; and wherein, whenthe mutation is E507K in combination with two or more further mutationsor the mutation is Q680R in combination with four or more furthermutations, at least one of the further mutations is at one of theselected amino acid positions; and when the mutation is I553V, this isnot in combination with D551S.

Taq DNA polymerase mutants may be provided with increased resistance toSYBR Green I dye present in PCR mixture as well as mutants able toperform DNA amplification faster and/or more efficiently as compared tothe wild type enzyme. One set of mutant variants of polymeraseoutperforms the wild type enzyme in PCR assays which contain SYBR GreenI fluorescent dye in reaction mixture. Another set of mutants is usefulin different PCR applications with shorter DNA elongation times ascompared to the ones typically required for the wild type Taq DNApolymerase. A third set of mutants exhibit increased resistance toblood, SDS, GuHCl and heparin inhibition and may be used in direct DNAamplification from blood samples (at blood concentrations, which areinhibitory to wild type Taq DNA polymerase) or from unpurified/partiallypurified DNA samples in different lysis buffers.

The wild type Taq DNA polymerase amino acid sequence is shown in FIG. 1.The numbering system used in the present application is based on thissequence. A DNA polymerase mutant according to the present inventioncomprises the wild type sequence with a mutation at one of the indicatedselected amino acid positions or at a plurality of the indicatedselected amino acid positions. Where there is a mutation at one or moreof the indicated selected amino acid positions it is also possible forthere to be one or more further mutations at other positions in the wildtype sequence. The number of these further mutations and the position ofany such further mutation is such that the properties of increasedpolymerase speed, increased affinity to DNA substrate and increasedresistance to a DNA polymerase inhibitor conferred by mutation at theindicated selected amino acid positions are not impaired. Such furthermutations are preferably conservative mutations. In a preferredembodiment mutations of the wild type sequence occur only in theindicated selected amino acid positions. It is also possible for thereto be additions to the amino acid sequence, for example at one or bothends of the sequence, without substantially affecting the activity ofthe polymerase.

Advantageously, the number of mutations at the one or more selectedamino acid positions is limited, for example to no more than three ofthe selected amino acid positions. It has surprisingly been found that arelatively low number of mutations can give rise to advantageousproperties of a mutant polymerase. In one arrangement, the amino acidsequence has a mutation at only one of the selected amino acidpositions. In another arrangement, the amino acid sequence has amutation at only two of the selected amino acid positions. By providingDNA polymerase mutants with a limited number of mutations in the primarystructure it is thought that the tertiary, three dimensional structureof the polymerase is not altered significantly. Mutants according to theinvention can exhibit relative to wild type DNA polymerase one or moreof the advantageous properties of increased polymerase speed, increasedaffinity to DNA substrate and increased resistance to a DNA polymeraseinhibitor.

In one aspect of the invention, the DNA polymerase mutant exhibitsincreased polymerase speed relative to wild type DNA polymerase. SuchDNA polymerase mutants include those with a mutation at one or more ofthe following selected amino acid positions: E189K, E230K, E507K, H28R,L30R, F73V, H75R, E76A, E76G, E76K, E90K, K206R, A439T, D452N, G504S,D551N, I553V, H676R, D732G, E734G, F749V. It is preferred that the DNApolymerase mutant exhibits an increased polymerase speed which is atleast 1.5 times faster than wild type DNA polymerase, preferably atleast three times and more preferably at least 12 times faster.Polymerase speed may be measured by performing PCR on phage lambda DNAsuch as a 1825 bp fragment of phage lambda DNA. Alternatively, thepolymerase speed may be measured by performing PCR on human genomic DNAsuch as, for example, on a 2.5 kbp fragment. The PCR buffer used forsuch assays may be based either on KCl or on ammonium sulfate.Quantitative analysis of PCR products may be performed using agarose gelelectrophoresis, generally with 1% gels. Further details of typicalmeasurements are presented in Example 2 below.

In a further aspect, the DNA polymerase mutant according to theinvention exhibits increased affinity to DNA substrate, relative to wildtype DNA polymerase. The DNA polymerase mutant preferably includes amutation at one or more of the following selected amino acid positions:E189K, E230K, E507K, H75R, E315K, A348V, L351F, L552R, D578N. The DNApolymerase mutant may include double mutations wherein the selectedamino acid positions are preferably selected from: H28R+E507K,H28R+Q680R, E507K+Q680R, L552R+Q680R, E230K+E507K, E189K+E507K,E315K+E507K, E230K+E315K, E507K+L552R.

Increased affinity to DNA substrate is generally expressed in terms ofthe dissociation constant Kd, which may typically be measured for a DNAoligoduplex substrate for example using an electrophoretic shiftmobility assay following incubation in a suitable buffer such as 40 mMTris, 20 mM acetic acid, 1 mM EDTA at pH8.4, in the presence of 10% v/vglycerol at 4° C. for 30 mins.

The Kd of wild type Taq DNA polymerase under these conditions isgenerally in the range 1.71 to 3.97 nM. Thus, a value of Kd below 1.71nM denotes increased affinity to the DNA oligoduplex substrate relativeto the wild type polymerase. It is preferred that the Kd for the mutantpolymerase is no more than 1 nM and is typically in the range 0.14 to 1nM.

In a further aspect, the DNA polymerase mutant according to theinvention exhibits increased resistance to a DNA polymerase inhibitor.The DNA polymerase inhibitor may be selected from SYBR Green I dye,blood, SDS, guanadinium salts and heparin. Such DNA polymerase mutantspreferably include a mutation at one or more of the following selectedamino acid positions: E189K, E230K, E507K, H28R, L30R, G38R, H75R, E76A,E76G, E76K, E90K, E315K, A439T, D452N, G504S, E507A, D551N, L552R,I553V, D578N, H676R, Q680R, D732G, E734G, E734K. The DNA polymerasemutants may include a double mutation at the following selected aminoacid positions: H28R+E507K, H28R+Q680R, E507K+Q680R, L552R+Q680R,E230K+E507K, E189K+E507K, E315K+E507K, E230K+E315K, E507K+L552R. SuchDNA polymerase mutants may exhibit both increased resistance to a DNApolymerase inhibitor and exhibit an increased affinity to a DNAsubstrate relative to wild type DNA polymerase.

Preferably, the Kd of a DNA polymerase mutant exhibiting increasedresistance to a DNA polymerase inhibitor for a DNA oligoduplex substrateis no more than 10 nM in the presence of SYBR Green I dye at aconcentration of approximately 0.4 μm. This is typically measured asdescribed above by electrophoretic shift mobility assay followingincubation in a suitable buffer such as 40 mM Tris, 20 mM acetic acid, 1mM EDTA at pH8.4, in the presence of 10% v/v glycerol at 4° C. for 30mins.

In one preferred arrangement according to the invention the DNApolymerase mutant has mutations at one or more of the amino acidpositions E189K, E230K and E507K. Mutations at two or three of thesepositions give rise to a DNA polymerase mutant which has both increasedaffinity to DNA substrate and increased resistance to SYBR Green I dye.

In a further aspect, the present invention provides a kit for nucleicacid amplification, such as PCR, which comprises a DNA polymerase mutantas described herein together with one or more reagents for a DNAsynthesis reaction. Such reagents include appropriate buffers, primersand nucleotides suitable for the nucleic acid amplification reaction.

The invention further provides a process for the production of a DNApolymerase mutant as described herein. The process comprises:

-   -   (1) subjecting a polynucleotide encoding a DNA polymerase to        error-prone PCR to generate a mutant library comprising an array        of differently-mutated polynucleotides;    -   (2) screening the mutant library for increased polymerase speed,        increased polymerase affinity to DNA substrate or increase        resistance to a DNA polymerase inhibitor;    -   (3) selecting one or more mutant DNA polymerases from screening        step 2; and    -   (4) repeating steps 1 to 3 until a final DNA polymerase mutant        is obtained.

According to this process, the mutant library produced by error-pronePCR in step (1) comprises an array of polynucleotides at least some ofwhich incorporate one or more mutations. On the basis that the mutationsare generated in an essentially random way, some polynucleotides willencode DNA polymerases which are non-functional, some will encode DNApolymerases which have essentially normal function and others willencode DNA polymerases with properties which are either superior orinferior to the wild type DNA polymerase. Screening step (2) maytypically be performed on one or more members of the library so as toidentify the desired characteristics of polymerases encoded by the oneor more members of the library. Following selection step (3) apolynucleotide encoding one or more selected DNA polymerases issubjected once again to error-prone PCR in accordance with step (1) andthe process is repeated until such time as a suitable mutant polymeraseis obtained through screening and selection. This process of directedevolution is described in further detail below.

DETAILED DESCRIPTION OF INVENTION

The invention will now be described in further detail, by way of exampleonly, with reference to the accompanying drawings, in which:

FIG. 1 shows the amino acid sequence of wild type Taq DNA polymerase;

FIG. 2 shows the results of gel electrophoresis following PCR of a 250bp amplicon performed with wild type polymerase and a pool of mutantpolymerases at different SYBR Green I concentrations;

FIG. 3 shows the sequencing results of Taq DNA polymerase mutantsselected after high throughput screening for SYBR Green I resistance;

FIG. 4 shows the frequency of mutations found during high throughputscreening of Taq DNA polymerase for SYBR Green I resistance;

FIG. 5 shows the results of gel electrophoresis following PCR of a 200bp amplicon comparing wild type polymerase with polymerases of theinvention;

FIG. 6 shows the results of gel electrophoresis following PCR of a 500bp amplicon at different SYBR Green I concentrations using wild typeenzymes or enzymes according to the invention;

FIG. 7 shows the results of polyacrylamide gel electrophoresis in anelectrophoretic mobility shift assay for wild type polymerase andpolymerases according to the invention;

FIG. 8 shows the results of polyacrylamide gel electrophoresis in anelectrophoretic mobility shift assay for wild type polymerase andpolymerases according to the invention in the presence of SYBR Green Idye;

FIG. 9 shows the sequence of plasmid1 used as a PCR target for DNAamplification;

FIG. 10 shows the sequencing results of Taq DNA polymerase mutantsselected after first high throughput screening of Taq DNA polymeraselibrary for shorter amplification and annealing times;

FIG. 11 shows the frequency of mutations found during first highthroughput screening of Taq DNA polymerase library for shorteramplification and annealing times;

FIG. 12 shows the sequencing results of Taq DNA polymerase mutantsselected after the second high throughput screening of Taq DNApolymerase library for shorter amplification and annealing times;

FIG. 13 shows the frequency of mutations found during the second highthroughput screening of Taq DNA polymerase library for shorteramplification and annealing times;

FIG. 14 shows the results of gel electrophoresis following PCR of a 1825bp amplicon using various polymerases in the presence of ammoniumsulfate or potassium chloride;

FIG. 15 shows the results of agarose gel electrophoresis following PCRon a 2.5 kbp amplicon with various polymerases in the presence ofammonium sulfate or potassium chloride;

FIG. 16 shows the results of gel electrophoresis following PCR of a1.825 kbp amplicon in the presence of blood comparing commercial andwild type polymerase with polymerases according to the invention;

FIG. 17 shows the results of gel electrophoresis following PCR of a1.825 kbp amplicon performed in the presence of SDS comparing commercialTaq polymerase and wild type Taq polymerase with polymerases accordingto the invention;

FIG. 18 shows the results of gel electrophoresis following PCR of a1.825 kbp amplicon performed in the presence of guanidiniumhydrochloride comparing commercial Taq polymerase and wild type Taqpolymerase with polymerases according to the invention; and

FIG. 19 shows the results of gel electrophoresis following PCR of a1.825 kbp amplicon performed in the presence of heparin comparingcommercial Taq polymerase and wild type Taq polymerase with polymerasesaccording to the invention.

EXAMPLE 1 Mutant Taq DNA Polymerase Library Screening for IncreasedResistance to SYBR Green I Dye

Taq DNA polymerase is widely used in qPCR because it is a robust andefficient enzyme, which has 5′-3′ exonuclease activity (required toactivate Taqman probe), no 3′-5′ exonuclease activity (no degradation ofPCR primers) and is not sensitive to dUTP used to avoid contamination inqPCR master mixes. SYBR Green I intercalating dye used in qPCR inhibitsTaq DNA polymerse (Nath et al., 2000) and can decrease PCR efficiencyand sensitivity. In some cases problem can be solved by adjusting buffercomposition, reaction conditions and/or using higher amounts of Taq DNApolymerase.

In order to select for Taq DNA polymerase mutants with increasedresistance to SYBR Green I we have performed directed evolution of TaqDNA polymerase. The amino acids sequence of parental wild type Taq DNApolymerase used for mutagenesis is given in FIG. 1. The initial libraryof genes (L0) coding for mutant Taq DNA polymerases was generated byerror-prone PCR using a modified protocol described by Zaccolo et al.(Zaccolo et al., 1996). Quality of the library was checked by sequencingof 8 randomly picked clones. One clone had deletion, which resulted inframeshift of coding sequence. Other 7 clones had from 2 to 6 nucleotidesubstitutions per gene. The ratio of transitions to transversions was2.4:1. As a result mutant polymerases had from 1 to 5 amino acidschanges or on the average 2.85 mutations per gene.

SYBR Green I dye 10,000× stock solution was obtained from Invitrogen andwas used in our experiments. Several rounds of high-throughput screeningwere performed for the expressed polymerase ability to perform PCR atincreasing concentrations of SYBR Green I (0.6×-2.5×). After the severalscreening rounds 37 random clones of individual mutants and the pool ofall selected mutants were chosen for further investigation. Initiallythe pool of plasmids encoding selected polymerases was purified. Thenthe N-terminal (His)₆GlyAla tag was fused to PCR amplified Taqpolymerase genes. Pool of mutant enzymes and wt Taq polymerase with thesame affinity tag were expressed in E.coli cells and purified usingNi-NTA Superflow (Qiagen) chromatography. In order to check theefficiency of our screening we have tested wt Taq polymerase and pool ofmutants for ability to perform PCR at different SYBR Green Iconcentrations (FIG. 2). In our particular (target/primer/buffer)amplification system Taq DNA polymerase typically can synthesize 250 bpPCR fragment in the presence of 0.2-0.5× SYBR Green I dye. Meanwhile thepool of mutant Taq DNA polymerases can generate the same 250 bp PCRfragment in the presence of at least 2 times higher concentration (1×)of SYBR Green I dye (FIG. 2). It is evident that in case of enzyme poolresistance to SYBR Green I inhibition is an average value and some ofthe individual enzymes from the pool should have higher resistances, andsome lower than the average resistance of the pool of polymerases.Different properties of selected mutant enzymes are determined byvarious mutations accumulated during the mutagenesis/screeningprocedure. Some mutations should be beneficial, some can besupplementary, neutral or even negative. Therefore it is critical tounderstand the nature of selected mutants and elucidate individualproperties of single amino acids changes. As a consequence 37 randomclones of individual positive hits were sequenced and analized (FIG. 3).Two clones (L6M1_(—)1, L6M4_(—)1) had stop codons and were excluded fromfurther analyzis. The number of amino acid changes in selected mutantsvaries from 1 to 8. On average there are 3.6 amino acids changes pergene. The frequency of all found mutations was calculated and is givenin FIG. 4. The most often mutated position in our selection wasglutamate 507 (E507K−11 mutants; E507A−3). There are 4 selected clones,which contain only single mutation of E507 amino acid (L6M8_(—)1−E507A;L6M40_(—)1, Taq_B3, Taq_A4−E507K) (FIG. 1). Other most frequentlymutated positions are H28 (H28R−6); F27 (F27L−3; F27S−1); K219(K219R−4); E230 (E230K−3; E230G−1); E76 (E76G−3) and E189 (E189K−3).

The general assumption is that most frequently mutated amino acids arethe most important and have the biggest impact on Taq DNA polymeraseresistance to SYBR Green I inhibition. In order to elucidate individualproperties of different mutations single and multiple mutants of Taqpolymerase were constructed (with addition of N-terminal (His)₆GlyAlatag for purification), expressed, partially purified and analyzed. Thewt and mutant Taq DNA polymerases were purified using two stepprocedure: initial denaturation of E. coli proteins for 15 minutes at75° C. and subsequent Ni-NTA affinity chromatography. As a result TaqDNA polymerase variants were typically purified to ˜80% homogeneityaccording to SDS-PAGE densitometry analysis. The activities of purifiedpolymerases were evaluated using standard polymerase unit definitionassay and if necessary (for example in PCR applications) equal amountsof polymerase units were used for analysis.

The ability of wt and individual mutants of Taq polymerase to performPCR at different SYBR Green I concentrations was tested using manydifferent target/primer/buffer systems. In this example we present twoPCR performed either on plasmid DNA (˜200 bp fragment) or on humangenomic DNA (˜500 bp fragment). Amplification is performed in thepresence of 0.2-5× concentration of SYBR Green I dye. The thresholdconcentration of SYBR Green I dye (at which full length DNA fragment isstill synthesized) is determined and is used to characterize enzyme ofinterest resistance to SYBR Green. The precise threshold value dependson particular target/primer/buffer system used for PCR, therefore it isvery important to have wt Taq DNA polymerase control reactions performedin parallel. Agarose gel electrophoresis pictures of typical experimentare shown in FIGS. 5 and 6. In both cases (amplification of 200 bp and500 bp DNA fragments) wt Taq DNA polymerase can synthesize PCR productat 0.5× concentration of SYBR Green I dye in reaction mixture. MeanwhileTaq DNA polymerase mutants tolerate substantially higher concentrationsof SYBR Green I dye in reaction mixture (E189K−1.5× (200 bp) and 2.5×(500 bp); E230K−2.5× (200 bp) and 2× (500 bp); E507K−2× (200 bp) and 3×(500 bp)). Summarized data of SYBR Green I dye inhibition in PCRexperiments are presented in Table 1. Performed PCR inhibitionexperiments allowed us to identify many individual mutations, whichincrease Taq DNA polymerase resistance to SYBR Green I dye and may beused in production of commercial enzymes and kits. Different mutantsincrease Taq polymerase resistance from 2 to 10 times (1-5×concentration of SYBRGreen I). Enzyme resistance to SYBR Green I dye isadditive (cumulative) and in most cases can be increased constructingdouble or triple mutants. For example, mutant E230K can tolerate 2-2.5×and E507K−2-3× concentration of SYBR Green I. Subsequently double mutantE230K+E507K can tolerate 3-4.5× and triple mutantE230K+E507K+E189K−3.5-5× concentration of SYBR Green I (Table 1). Manymore multiple mutant combinations were tested and found to haveincreased SYBR Green I resistance comparing to single mutants (Table 1).Additivity of SYBR Green resistance is very important feature, whichenables design of mutant polymerases with individual propertiesaccording to specific application requirements.

Bioinformatic analysis of selected Taq DNA polymerase mutants withincreased resistance to SYBR Green I dye revealed that in most caseschanges were associated with amino acids which eliminate negative charge(E76G, E507A, D578N, D732G), add positive charge (H28R, G38R, L552R,H676R, Q680R) or change negative charge to positive one (E90K, E189K,E230K, E315K, E507K). In all cases mutant polymerases acquired highertotal positive charge and either could less interact with positivelycharged SYBR Green I dye or should have increased affinity to negativelycharged substrate (DNA). In both cases such enzymes should become moreresistant to SYBR Green I inhibition during PCR. If this hypothesis iscorrect, then mutations scope could be broadened by using similarsubstitutions and constructing mutants with increased positive charge(amino acids change X=>K, R) or decreased negative charge network (aminoacids change D, E=>X). In order to test the polymerase affinity to DNAwe have measured dissociation constant (Kd) value of protein-DNAinteraction using electrophoretic mobility shift assay (EMSA). Wild typeTaq DNA polymerase and mutants Kd were measured directly (without SYBRGreen I) and with SYBR Green I. Polyacrylamid gel electrophoresispictures of typical experiment are shown in FIGS. 7 and 8. Calculated Kdvalues for wt Taq DNA polymerase and different mutant variants aresummarized in the Table 2. The Kd of wt Taq DNA polymerase and DNAoligoduplex complex was obtained to be in the range of 1.71-3.97 nMwithout SYBR Green I dye and in the range of 6.17-9.39 nM with SYBRGreen I (0.2×). Meanwhile most mutant variants have Kd below 1.0 nMwithout SYBR Green I (E189K, E230K, E315K, E507K, L552R, D578N,H28R+E507K, H28R+Q680R, E507K+Q680R, L552R+Q680R, E230K+E507K,E189K+E507K, E315K+E507K, E230K+E315K, E507K+L552R, E189K+E230K+E507K)and <10 nM Kd in the presence of SYBR Green I. It is also important tostress that EMSA measurements performed at 0.1 nM concentration ofoligoduplex are good to calculate Kd values above 1 nM and give onlyapproximate results for Kd values below 1 nM. Consequently Kd valuesdetermined for mutant Taq DNA polymerases to be in the range of 0.14-1nM can be much lower. Overall data confirm the statement, that mutationsof interest, which were identified, possess 5-10 times increasedaffinity to DNA substrate (E189K, E230K, E315K, E507K, L552R, D578N,H28R+E507K, L552R+Q680R, E230K+E507K, E189K+E507K, E315K+E507K,E230K+E315K, E507K+L552R, E189K+E230K+E507K). It is very likely, thatSYBR Green I, present in PCR, binds to DNA target, hinders polymerasebinding to substrate and in such a way decreases the efficiency of PCR.Consequently mutant polymerases with increased affinity are moreresistant to SYBR Green I inhibition. The Kd of some mutant polymerases(E189K, E230K, E315K, E507K, L552R, D578N, H28R+E507K, H28R+Q680R,E507K+Q680R, L552R+Q680R, E315K+E507K, E230K+E315K, E507K+L552R,E189K+E230K+E507K) in the presence of SYBR Green I (0.2×) is increasedto the level of wt Taq DNA polymerase Kd without SYBR Green I and isbelow 5 nM (Table 2). Diminished Kd of mutant polymerases and DNA/DNAsubstrate complex confirms the hypothesis, that positive charges(accumulated during the screening of Taq DNA polymerase for SYBR Green Idye resistance) increase the affinity of enzyme to negatively chargedDNA and in such a way neutralize the inhibitory effect of positivelycharged SYBR Green I dye. Taq DNA polymerases with increased affinitycould be very useful in many different PCR and qPCR applications sincein some cases increased affinity can lead to increased enzymeprocessivity, polymerization velocity, resistance to differentinhibitors, more sensitive PCR, etc.

Methods and Materials Polymerase Purification

The expression plasmid coding for the mutant or wt Taq DNA polymerasevariants fused to the N-terminal (His)₆GlyAla tag was expressed in E.coli cells. The E. coli cells were harvested by centrifugation at 4° C.(5000 rpm for 10 min, Beckman J2-21 centrifuge, JA-10 rotor) andresuspended in buffer A (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole and10 mM 2-mercaptoethanol, pH 8.0) with 1 mMphenylmethanesulphonyl-fluoride (Sigma). After sonication on ice (7.5min), samples were centrifugated at 16 170 g for 20 min (Eppendorf5417R). Next, the supernatant was heated at 75° C. for 15 min todenature most of the E. coli mezofilic proteins.

Precipitated proteins were removed by centrifugation (at 16 170 g for 20min) and the supernatant was loaded onto a Ni-NTA Superflow (Qiagen)minicolumn. To remove unspecifically bound proteins, the minicolumn waswashed with buffer B (50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, 10 mM2-mercaptoethanol and 0.1% Triton X-100, pH 8.0); the polymerase waseluted with buffer C (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole, 10mM 2-mercaptoethanol and 0.1% Triton X-100, pH 8.0). Polymerase wasdialysed against storage buffer (20 mM Tris-HCl (pH 8.0), 1 mM DTT, 0.1mM EDTA, 100 mM KCl, 0.5% (v/v) Nonidet P40, 0.5% (v/v) Tween 20 and 50%(v/v) glycerol) and stored at −20° C.

Polymerase Unit Definition Assay

The DNA polymerase activity of purified mutant Taq DNA polymerases wasmeasured according to the following protocol. The enzyme was incubatedin a reaction mixture (50 μl) consisting of 67 mM Tris-HCI (pH 8.8), 6.7mM MgCl₂, 50 mM NaCl, 1 mM DTT, 0.1 mg/ml BSA, 200 μM of each of dATP,dCTP, dTTP and dGTP, 0.4 MBq/ml of [methyl-³H]thymidine 5′-triphosphate(Amersham), and 250 μg/ml of activated salmon sperm DNA at 70° C. for 30min. The reaction was stopped on ice, and an aliquot was spotted onto aDE-81 filter-paper disc. The disc was dried on a heat block, washed in7.5% sodium phosphate buffer for 5 minutes 3 times and once in 70%ethanol for 2 minutes, and then dried again. The incorporatedradioactivity on the dried filter-paper disc was counted using a BeckmanLS-1801 scintillation counter. One unit of Taq DNA polymerase catalyzesthe incorporation of 10 nmol of deoxyribonucleotides into apolynucleotide fraction (adsorbed on DE-81) in 30 min at 70° C.

Mutagenic PCR

Mutant Taq DNA polymerase gene variants were constructed using modifiederror-prone PCR protocol described by Zaccolo (Zaccolo et al., 1996).Briefly, a PCR comprising 75 mM Tris-HCl (pH 8.8 at 25° C.), 20 mM(NH₄)₂SO₄, 0.01% (v/v) Tween 20, 10 ng template DNA forward and reverseprimers (0.5 μM each), dNTPs (200 μM each), 0.4 μM dPTP (TriLinkBioTechnologies), 10 μM 8-oxo-dGTP (TriLink BioTechnologies), 1.5 mMMgCl₂ and 9.75 u of Taq polymerase in a total volume of 390 μL wascarried out with the thermal profile 2 min at 94° C. followed by 30cycles of 30 s 94° C., 30 s 50° C., 2 min 40 s 72° C. and finished with10 min at 72° C. Amplified PCR product was digested with appropriaterestriction endonucleases and cloned into an expression vector using T4DNA ligase.

Mutant Taq DNA Polymerases are More Tolerant to SYBR Green I Dye in PCR

10000× SYBR Green I dye solution was obtained from Invitrogen (S7567)and stored in small aliquots at −20° C. Fresh serial dilutions (innuclease-free water) of SYBR Green I dye stock solution were used inPCR. A typical SYBR Green I dye solution at a dilution of 0.2× isestimated to have a concentration of 0.4 μM (Gudnason et al 2007, Zipperet al 2004).

Amplification of 250 bp Bacterial Plasmid DNA Target with Pool of MutantTaq Polymerases

PCR mixtures comprising 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 0.08% (v/v)Nonidet P40, dNTPs (200 μM each), 1.5 mM MgCl₂, 0.5 μM each of primer 1and 2 (Table 3), 6 ng plasmid1 DNA (FIG. 9), 0.5 u of polymerase andvarious amounts of SYBR Green I dye (at the final concentration of 0,0.2, 0.5, 1, 1.5, 2, 4×) in a total volume of 20 μL were subjected tothe following thermocycling conditions: 2 min at 94° C. followed by 30cycles of 30 s 94° C., 30 s 50° C., 20 s 72° C. PCR products wereanalyzed in 2% agarose gel electrophoresis.

Amplification of 200 bp Bacterial Plasmid DNA Target with Mutant TaqPolymerases

PCR mixtures comprising 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 0.08% (v/v)Nonidet P40, dNTPs (200 μM each), 1.5 mM MgCl₂, 0.5 μM each of primer 3and 4 (Table 3), 6 ng plasmid1 DNA (FIG. 9), 0.5 u of polymerase andvarious amounts of SYBR Green I dye (at the final concentration of 0,0.2, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5×) in a total volume of 20μL were subjected to the following thermocycling conditions: 3 min at94° C. followed by 30 cycles of 30 s 94° C., 30 s 50° C., 15 s 72° C.PCR products were analyzed in 1-2% agarose gel electrophoresis.

Amplification of 500 bp Human Genomic DNA Target with Mutant TaqPolymerases

PCR mixtures (20 μL) containing: 10 mM Tris-HCl (pH 8.8), 50 mM KCl,0.08% (v/v) Nonidet P40, dNTPs (200 μM each), 1.5 mM MgCl₂, 0.5 μM eachof primer 5 and 6 (Table 3), 40 ng of human genomic DNA, 0.5 u ofpolymerase and various amounts of SYBR Green I dye (at the finalconcentration of 0, 0.2, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5×) weresubjected to the following thermocycling conditions: 3 min at 95° C.followed by 35 cycles of 30 s 95° C., 30 s 60° C., 30 s 72° C. PCRproducts were analyzed in 1% agarose gel electrophoresis.

Increased Affinity of Mutant Taq DNA Polymerases for Primer-Template DNAPreparation of Radioactively Labeled Probe for Electrophoretic MobilityShift Assay

Radioactively labeled probe for electrophoretic mobility shift assay wasprepared as follows. A single-stranded 24-mer oligonucleotide 1 wasradioactively labeled at 5′-termini with polynucleotide kinase (PNK).Briefly, reaction mixture containing 1× PNK buffer A (50 mM Tris-HCl (pH7.6), 10 mM MgCl₂, 5 mM DTT, 0.1 mM spermidine), 1 μM oligonucletide 1,1 μM [γ-³³P]-ATP (Hartmann Analytic) and 0.5 u/μl PNK was incubated at37° C. for 30 min, then PNK was inactivated by heating the sample at 70°C. for 10 min. A dsDNA probe for electrophoretic mobility shift assayswas prepared by annealing radioactively labeled oligonucleotide 1 to asingle-stranded 44-mer oligonucleotide 2 as follows. A mixtureconsisting of 75 mM Tris-HCl (pH 8.8), 20 mM (NH₄)₂SO₄, 0.01% (v/v)Tween 20, 2 mM MgCl₂, 20 nM of unpurified radioactively labeledoligonucleotide 1 and 25 nM of oligonucleotide 2 was incubated at 94° C.for 4 min, then the sample was transferred to the glass with pre-boiledwater and left to cool slowly overnight.

Electrophoretic Mobility Shift Assay

Serial dilutions of mutant Taq polymerase were prepared in polymerasestorage buffer: 20 mM Tris-HCl (pH 8.0), 1 mM DTT, 0.1 mM EDTA, 100 mMKCl, 0.5% (v/v) Nonidet P40, 0.5% (v/v) Tween 20 and 50% (v/v) glycerol.Mutant Taq polymerase at various concentrations (0.25, 0.5, 1, 2.5, 5,10, 25, 50, and 100 nM) was incubated with 0.1 nM radioactively labeleddsDNA probe (see above) in 1× TAE buffer (40 mM Tris, 20 mM acetic acid,1 mM EDTA, pH 8.4) with 10% (vol/vol) glycerol and without or with SybrGreen I dye (0.2× final concentration) at +4° C. for 30 min. 5 μl ofsamples were run on a native 12% polyacrylamide gel to separate theprotein-DNA complex from the free DNA. Gels were dried and exposed tostorage phosphor screens (FujiFilm). Screens were scanned withphosphorimager scanner (Typhoon) at resolution 100. The concentration ofpolymerase-DNA complex was determined from scanned gels using TotalLab100; the dissociation constant of polymerase-DNA complex K_(d) (nM) wascalculated with GraphPad using the following equation:

[ES]=([E] ₀ +[S] ₀ +K _(d) −sqrt(sqr([E] ₀ +[S] ₀ +K _(d))−4[S] ₀ [E]₀))/2,

where [ES] is the concentration of formed polymerase-DNA complex (nM),[E]₀ is the initial concentration of polymerase (nM), [S]₀−the initialconcentration of DNA substrate (0.1 nM).

EXAMPLE 2 Mutant Taq DNA Polymerase Library Screening for IncreasedAmplification Speed

PCR and qPCR applications are widely used in almost all molecularbiology, biochemistry and clinical diagnostic laboratories over theworld. Faster PCR applications are highly desirable due to the fact thatthey decrease time spent for DNA amplification, required machine workingtime and increase the efficiency of analytical procedure. Fast PCRmachines are already available on the market and now the limiting stepis the enzyme suitable for fast applications. Taq DNA polymerase, whichis widely used in PCR and qPCR, was subjected to in vitro evolutionaiming to increase its amplification speed and decrease time requiredfor elongation step during PCR. Two different libraries of Taq DNApolymerase were used for high-throughput screening in this example.

1^(st) Screening

The same mutant Taq DNA polymerase library (L0) as in Example 1 was usedfor selection of improved Taq DNA polymerase variants. Several rounds ofhigh-throughput screening were performed testing expressed polymeraseability to perform PCR using shorter and shorter amplification andannealing times with every screening round. Subsequently 43 randomclones of individual mutants were sequenced and analized (FIG. 10). Twoclones (L8-10, L8-42) had stop codons and were excluded from furtheranalyzis. The number of amino acid changes in selected mutants variesfrom 2 to 12. On average there are 5.4 amino acids changes per gene. Thefrequency of all identified mutations was calculated and is given inFIG. 11. The most often mutated positions in this selection wereglutamate 230 (E230K−33 mutants), leucine 30 (L30P−21 mutants; L30R−10;L30Q−1), aspartate 452 (D452N−18 mutants), glycine 504 (G504S−15mutants), leucine 311 (L311F−4 mutants; L311R−1), glutamate 507 (E507A−3mutants; E507G−2), glutamate 189 (E189K−4 mutants). There are 2identical selected clones (L8-9, L8-39), which specifically contain only4 most frequent mutations (L30P, E230K, D452N, G504S).

2^(nd) Screening

An additional screening of Taq DNA polymerase for mutant variants ableto perform PCR at shorter amplification and annealing times wasperformed using library (L3) with increased mutational load. The L3library was also generated by error-prone PCR using a modified protocoldescribed by Zaccolo et al. (Zaccolo et al., 1996). Quality of thelibrary was checked by sequencing of 9 randomly picked clones. Twoclones had deletions, which resulted in frameshift of coding sequence.Other 7 clones had from 2 to 9 nucleotide substitutions per gene. Theratio of transitions to transversions was 6:1. As a result mutantpolymerases had from 1 to 5 amino acids changes or on the average 3mutations per gene. Several rounds of high-throughput screening wereperformed testing expressed polymerase ability to perform PCR usingshorter and shorter amplification and annealing times with everyscreening round. Subsequently 42 random clones of individual mutantsafter the screening were sequenced. Two clones had deletions/insertions,three clones had truncated N terminus and were omitted from analyzis(FIG. 12). The number of amino acid changes in selected mutants variesfrom 2 to 10. On average there are 5.4 amino acids changes per gene. Thefrequency of all found mutations was calculated and is given in FIG. 13.The most often mutated positions in this selection were phenylalanine 73(F73V−3 mutants; F73S−1; F73L−1), aspartate 144 (D144G−4 mutants;D144N−1), lysine 206 (K206R−5 mutants), leucine 30 (L30P−3 mutants),histidine 75 (H75R−3 mutants), glutamate 90 (E90G−3 mutants), lysine 143(K143E−2 mutants; K143R−1), leucine 351 (L351F−3 mutants), glutamate 397(E397K−2 mutants; E397G−1), alanine 439 (A439T−3 mutants).

Mutant Analysis

The general assumption is that most frequently mutated amino acids foundduring the screening are the most important and have the biggest impacton Taq DNA polymerase properties. Site specific mutagenesis was used toconstruct novel polymerase mutants, by introducing mutation most oftenlyfound in our selective enrichment procedure and which were not describedelsewhere in the literature. In order to elucidate individual propertiesof different mutations single mutants of Taq polymerase were constructed(with addition of N-terminal (His)₆GlyAla tag for purification),expressed, partially purified and analyzed. The wt and mutant Taq DNApolymerases were purified using two step procedure: initial denaturationof E. coli proteins for 15 minutes at 75° C. and subsequent Ni-NTAaffinity chromatography. As a result Taq DNA polymerase variants weretypically purified to ˜80% homogeneity according to SDS-PAGEdensitometry analysis. The activities of purified polymerases wereevaluated using standard polymerase unit definition assay and, ifnecessary (for example in PCR applications), equal amounts of polymeraseunits were used for analysis. The ability of wt and individual mutantsof Taq polymerase to perform PCR using shorter amplification andannealing times was tested using few different target/primer/buffersystems. Four PCR were performed either on phage lambda DNA (1825 bpfragment) or on human genomic DNA (˜2.5 kbp fragment) using PCR bufferbased either on KCl or on (NH₄)₂SO₄ (see methods and materials).Amplification was performed using three different in length (Normal,Fast, Very fast) cycling conditions. In all cases wt Taq DNA polymerasewith His tag purified in similar way was used as a control. Agarose gelelectrophoresis pictures of typical experiment are shown in FIGS. 14 and15. Phage lambda DNA amplification (1825 bp fragment) was performedusing ˜30 s/kb (normal), ˜10 s/kb (fast) and 2.5 s/kb (very fast)extension rates. Recommended extension rate for Taq DNA polymerase is30-60 s/kb. In this test PCR wt Taq DNA polymerase used as a control wasable to amplify 1825 bp DNA fragment only under normal (˜30 s/kb)cycling conditions (FIG. 14). Mutant enzymes (point mutants) identifiedin our screening were able to amplify target under fast ˜10 s/kb (L30R;E230K; D452N; G504S; E507K; E189K) and even very fast ˜2.5 s/kb (L30R;E230K; E507K; E189K) cycling conditions (FIG. 14). Human genomic DNAamplification (˜2.5 kbp fragment) was performed using ˜50 s/kb (normal),˜25 s/kb (fast) and 12 s/kb (very fast) extension rates. In this testPCR wt Taq DNA polymerase used as a control has synthesized only minoramount of 2.5 kbp DNA fragment even under normal (˜50 s/kb) cyclingconditions (FIG. 15). Meanwhile mutant enzymes (point mutants)identified in our screening were able to amplify target under fast ˜25s/kb and even very fast ˜12 s/kb (E189K; E230K; E507K) cyclingconditions in (NH₄)₂SO₄ based buffer (FIG. 15A). Using KCl based buffersome mutant enzymes (point mutants) were able to amplify target underfast ˜25 s/kb (E230K; E507K) and even very fast ˜12 s/kb (E230K; E507K)cycling conditions (FIG. 15B). Many more point mutants of Taq DNApolymerase identified during our screenings were tested in the same typePCR assay under normal, fast and very fast cycling conditions.Summarized data on all PCR are given in Table 4. The Taq mutantconsidered to be fast if it was able to amplify both targets from phagelambda and human genomic DNA under fast cycling conditions at least inone buffer system (with (NH₄)₂SO₄ or with KCl): E76G; E76A; D551N;I553V; D732G; F73V; H75R; K206R; A439T; F749V; D452N; G504S (Table 4).The Taq mutant considered to be very fast if it was able to amplify bothtargets from phage lambda and human genomic DNA under very fast cyclingconditions at least in one buffer system (with (NH₄)₂SO₄ or with KCl):E90K; E189K; E230K; E507K; H676R; H28R; E76K; E734K; L30R (Table 4). Twohigh-throughput screenings using Taq mutants libraries L0 or L3 wereperformed in this example. The frequencies of all found mutations arecalculated and given in FIGS. 11 and 13. As in Example 1 most of mutantseither possess eliminated negative charge (D452N, E507G, E507A, D144G,D144N, E90G, E397G), added positive charge (L30R, L311R, H75R) orchanged negative charge to positive one (E230K, E189K, E397K).Consequently mutant polymerases became more positively charged and couldhave increased affinity to negatively charged substrate (DNA). In orderto test the polymerase affinity to DNA we have measured dissociationconstant (Kd) value of protein-DNA interaction using electrophoreticmobility shift assay (EMSA). Calculated Kd values for wt Taq DNApolymerase and different mutant variants are summarized in the Table 5.The Kd of wt Taq DNA polymerase and DNA oligoduplex complex was obtainedto be in the range of 1.71-3.97 nM (Table 2). In addition to alreadypreviously identified mutants with increased affinity Kd<1 nM (E230K,E189K) we have found, that mutants A348V, H75R and L351F have Kd<1 nM(Table 1) and should be attributed to the group of high affinity Taqmutants described in Example 1 (Table 2).

Mutants of Taq DNA polymerase able to perform PCR under fast or veryfast conditions could be used in fast PCR and qPCR applications, savinginstrument and researcher time, increasing laboratory throughput volume.

Methods and Materials Polymerase Purification

The same as in Example 1.

Polymerase Unit Definition Assay

The same as in Example 1.

Mutagenic PCR

The same as in Example 1.

Mutant Taq DNA Polymerases Synthesize DNA Faster in End-Point PCR ThanWild-Type Taq

Amplification of 1825 bp Phage Lambda DNA Target with Mutant Taq DNAPolymerases

PCR mixtures comprising 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 0.08% (v/v)Nonidet P40, 1.5 mM MgCl₂ (buffer with KCl) or 75 mM Tris-HCl (pH 8.8),20 mM (NH₄)₂SO₄, 0.01% (v/v) Tween 20, 2 mM MgCl₂ (buffer with(NH₄)₂SO₄) and dNTPs (200 μM each), 0.5 μM each of primer 7 and 8 (Table3), 0.25 ng phage lambda DNA, 0.5 u of polymerase in a total volume of25 μL were subjected to the following three thermocycling conditions:

-   -   1) 5 min at 95° C. followed by 20 cycles of 30 s 95° C., 30 s        60° C., 60 s 72° C.;    -   2) 5 min at 95° C. followed by 20 cycles of 30 s 95° C., 10 s        60° C., 20 s 72° C.;    -   3) 5 min at 95° C. followed by 20 cycles of 30 s 95° C., 5 s 60°        C., 5 s 72° C.

PCR was performed on Eppendorf Mastercycler, using “BLOCK CONTROL”option, ramping slope 3°/s. PCR products were analyzed in 1% agarose gelelectrophoresis.

Amplification of 2.5 kb Human Genomic DNA Target with Mutant TaqPolymerases

PCR mixtures (25 μL) comprising 10 mM Tris-HCl (pH 8.8), 50 mM KCl,0.08% (v/v) Nonidet P40, 1.5 mM MgCl₂ (buffer with KCl) or 75 mMTris-HCl (pH 8.8), 20 mM (NH₄)₂SO₄, 0.01% (v/v) Tween 20, 2 mM MgCl₂(buffer with (NH₄)₂SO₄) and dNTPs (200 μM each), 0.5 μM each of primer 9and 10 (Table 3), 125 ng human genomic DNA (blood purified, using“Genomic DNA purification Kit” (K0512-Fermentas)), 0.5 u of polymerasewere subjected to the following thermocycling conditions:

-   -   1) 5 min at 95° C. followed by 30 cycles of 30 s 95° C., 30 s        65° C., 2 min 72° C.    -   2) 5 min at 95° C. followed by 30 cycles of 30 s 95° C., 20 s        65° C., 1 min 72° C.    -   3) 5 min at 95° C. followed by 30 cycles of 30 s 95° C., 10 s        60° C., 30 s 72° C.

PCR was performed on Eppendorf Mastercycler, using “BLOCK CONTROL”option, ramping slope 3°/s. PCR products were analyzed in 1% agarose gelelectrophoresis.

Increased Affinity of Mutant Taq DNA Polymerases for Primer-Template DNA

The same as in Example 1.

EXAMPLE 3 Mutants of Taq DNA Polymerase Resistant to Different PCRInhibitors

Mutant polymerases with increased resistances could be used in PCR andqPCR applications without target DNA purification step (directly afterthe lysis or after partial/simplified purification step). Consequentlywe have tested most interesting Taq DNA polymerase mutants identified inexample 1 and example 2 in PCR performed with various inhibitors knownto be incompatible with wt Taq DNA polymerase. PCR was performed onphage lambda DNA using Taq DNA polymerase (recombinant, Fermentas,#EP0404) and wt His-Taq as the controls. The set of Taq DNA polymerasesingle amino acid mutants (E189K; E230K; E507K; H28R; E90K; E76K; H676R;L30R; D452N; E734K; D732G; D551N; I553V; G504S; H75R; E76G; E76A; A348V;A439T; E734G) was tested for increased resistance to different PCRinhibitors (blood; SDS, GuHCl, heparin).

Typical picture of PCR inhibition with blood is given in FIG. 16. Underchosen PCR conditions neither commercial Taq, nor wt his-Taq were ableto synthesize 1.8 kbp DNA fragment even in the presence of 0.5% ofblood. Meanwhile mutant polymerases (E189K, E230K, E507K, H28R) wereable to perform PCR in the presence of 4-8% of blood.

Typical picture of PCR inhibition with SDS is given in FIG. 17. Underchosen PCR conditions commercial Taq and wt his-Taq synthesized specificDNA fragment in the presence of 0.0025% and 0.001% of SDS respectively.Meanwhile mutant polymerases (E189K, E230K, E507K) were able to performPCR in the presence of 0.005-0.0075% of SDS.

Typical picture of PCR inhibition with GuHCl is given in FIG. 18. Underchosen PCR conditions commercial Taq synthesized specific DNA fragmentin the presence of 20 mM of GuHCl. Another control enzyme wt his-Taq(should be used for direct comparison with Taq polymerase mutants) wasnot able to synthesize 1.8 kbp DNA fragment even in the presence of 10mM of GuHCl. Meanwhile mutant polymerases (E189K, E230K, E507K, H28R)were able to perform PCR in the presence of 40-70 mM of GuHCl.

Typical picture of PCR inhibition with heparin is given in FIG. 19.Under chosen PCR conditions commercial Taq and wt his-Taq synthesizedspecific DNA fragment in the presence of 0.0025 UPS and 0.001 UPSheparin (per 25 μl of PCR reaction) respectively. Meanwhile mutantpolymerases (D452N, D551N, G504S) were able to perform PCR in thepresence of 0.0062 UPS heparin (per 25 μl of PCR reaction).

Summarized data on all PCR inhibition experiments are given in Table 6.Tested Taq DNA polymerase mutants have different resistances to variousPCR inhibitors. Some mutants are more resistant to blood (E189K, E230K,E507K, H28R), some mutants are more resistant to SDS (E189K, E230K,E507K, E90K, E76K, H676R, L30R, D452N, E734K, D732G, D551N, I553V,G504S, H75R, E76G, E76A, A439T, E734G), some mutants are more resistantto GuHCL (E189K, E230K, E507K, H28R, E90K, E76K, H676R, L30R) and somemutants are more resistant to heparin (D452N, D551N, G504S) (Table 6).Most interesting are Taq mutants with the highest resistances to testedPCR inhibitors (except heparin) and increased amplification speed:E189K, E230K and E507K (Table 6 and Table 4).

Mutants of Taq DNA polymerase able to perform PCR in the presence ofdifferent inhibitors are very important and can be used foramplification of partially purified or unpurified DNA samples. Skippedor simplified nucleic acids purification step makes diagnostic procedurefaster, cheaper and more convenient for user. Different “direct PCR”kits from plants, tissues, blood, etc could be prepared andcommercialized on the basis of newly discovered mutations and theircombinations.

Methods and Materials The Measurements of wt Taq DNA Polymerase andMutant Enzymes Resistance to Different PCR Inhibitors

Amplification of 1825 bp Phage Lambda DNA Target with Mutant Taq DNAPolymerases

PCR mixtures comprising 50 mM Tricine (pH 8.8), 20 mM (NH₄)₂SO₄, 0.01%(v/v) Tween 20, 2.5 mM MgCl₂, dNTPs (200 μM each), 0.5 μM each of primer7 and 8 (Table 3), 0.25 ng phage lambda DNA, INHIBITOR X and 1 u ofpolymerase in a total volume of 25 μL were subjected to the followingthermocycling conditions: 5 min at 95° C. followed by 30 cycles of 20 s95° C., 25 s 60° C., 80 s 72° C. PCR was performed on EppendorfMastercycler epgradient S, PCR products were analyzed in 1% agarose gelelectrophoresis.

INHIBITOR X:

Fresh blood stabilized with sodium citrate—0%, 0.5%, 1%, 2%, 4%, 8%(v/v);

SDS (Sodium Dodecyl Sulphate, Amresco 0227-1 kg)—0%, 0.001%, 0.0025%,0.005%, 0.0075%, 0.015% (w/v);

GuHCl (Guanidine Hydrochloride, Roth 0037.1)—0 mM, 10 mM, 20 mM, 40 mM,70 mM, 100 mM;

Heparin (Sigma, H3125)—0, 0.001, 0.0025, 0.00625, 0.015625, 0.039 UPSheparin (per 25 ml of PCR reaction).

TABLE 1 The threshold concentrations of SYBR Green I dye (at which fulllength DNA fragment is still synthesized) determined for wt anddifferent mutants of Taq DNA polymerase. SYBR Green I stockconcentration is 10′000X and amplification was performed using 0.2-5Xconcentration of dye. Amplification of 200 bp Amplification of 500 bpfragment from fragment from human Mutant name plasmid DNA genomic DNA wtHis-Taq 0.5 0.2-0.5 H28R 1 1 G38R 1 1 E76G 1 n.d. E90K 1.5 1.5 E189K 1.52.5 E230K 2.5 2 E315K 1.5 1.5 E507A 2 1.5 E507K 2 3 L552R 2 1.5 D578N 11.5 H676R 1 1.5 Q680R 1 1 D732G 1 1 H28R + E507K 2.5 n.d. H28R + Q680R1.5 1.5 E507K + Q680R 2.5 n.d. L552R + Q680R 3.5 n.d. E230K + E507K 4.53 E189K + E507K 5 4 E315K + E507K 4 4 E230K + E315K 3 2.5 E507K + L552Rn.d. 3 E189K + E230K + E507K 5 3.5 n.d.—not determined

TABLE 2 The dissociation constants (Kd) of wt and mutant Taq DNApolymerases and oligoduplex substrate determined without and with (0.2X)SYBR Green I dye. EMSA measurements were performed at fixed 0.1 nMconcentration of oligoduplex and 0.25-100 nM concentration gradient ofpolymerase. Kd Mutant name Kd, nM (+0.2X SYBR Green I), nM wt His-Taq 1.71-3.97* 6.17-9.39 H28R 0.83-1.59 2.61-8.39 G38R 2.47 7.24 E90K 1.537.86 E189K 0.40 2.69 E230K 0.37 4.76 E315K 0.50 2.50 E507A 1.14 7.29E507K 0.18-0.19 3.91-4.22 L552R 0.53 1.88-7.49 D578N 0.70 2.66 H676R4.87 10.94  Q680R 3.18 9.61 D732G 1.82 8.10 H28R + E507K 0.25 4.71H28R + Q680R 0.95 2.17 E507K + Q680R 0.71 3.25 L552R + Q680R 0.29 2.63E230K + E507K 0.18 2.73-7.95 E189K + E507K 0.29 6.61 E315K + E507K 0.303.66 E230K + E315K 0.37 2.60 E507K + L552R 0.25 2.13 E189K + E230K +E507K 0.14 0.43 *the range of Kd values is given if more than oneexperiment was performed

TABLE 3 The oligonucleotide sequences used in Examples. Oligo-nucleotide Designation Sequence (5′ to 3′) Primer 1 agseqlGCGTTATCTCATAGACAAGG GC Primer 2 agseq2 GTAAGTTATTATCACATCCG GGTCPrimer 3 Pra1 AATGGCTAGCTGGAGCCAC Primer 4 Seq promTATCTCCTCAATAGCGGAGTCATC Primer 5 Forward CAAGGTCATCCATGACAACTTTG GAPDHPrimer 6 Reverse GTCCACCACCCTGTTGCTGTAG GAPDH Oligo- LA422TTTTAGCCGCTAGAGTCGACCTGC nucleotide 1 Oligo- LA424GGAGACAAGCTTGTATGCCTGCAGGT nucleotide 2 CGACTCTAGCGGCTAAAA Primer 7 L-16ATCCTGAACCCATTGACCTCCAAC Primer 8 L-19 ACTGAATCCCCGATCATCTATCGC Primer 9CAGCTCAGTGGTTTTCATTG GTTG Primer 10 CTGTGAGGCAGAGACAGCAGAGAC

TABLE 4 The summarized data on different PCR experiments performed undernormal (1), fast (2) and very fast (3) cycling conditions. PCR wasperformed on two different targets: phage lambda and human genomic DNAin two different buffers based either on (NH₄)₂SO₄ or on KCl.amplification of 1825 bp DNA fragment from amplification of 2.5 kbp DNAfragment from phage lambda DNA human genomic DNA with (NH₄)₂SO₄ with KClwith (NH₄)₂SO₄ with KCl Polymerase 1 2 3 1 2 3 1 2 3 1 2 3 Taq DNA pol.++ ++ + + Fermentas #EP0404 wt His-Taq ++ ++ + + Y24H ++ ++ ++ ++ T26P++ +++ + + F27S + +++ + + G38R +++ + +++ ++ ++ + E76G +++ ++ +++ + ++ +++ + E90K +++ ++ + ++ ++ ++ ++ ++ + nsp E189K +++ ++ + + + ++ ++ + +E230K +++ ++ + +++ ++ ++ +++ ++ + + + E507K +++ ++ + +++ ++ + +++ ++++ + + + D551N +++ ++ +++ +++ + +++ ++ +++ ++ I553V ++ + +++ + + ++ +++++ H676R +++ ++ +++ +++ ++ +++ ++ +++ ++ + D732G +++ ++ +++ ++ ++ +++ ++++ ++ H28P ++ +++ + + + H28R +++ +++ + +++ ++ + +++ ++ + +++ ++ F73V ++++ + + + + H75R +++ ++ +++ ++ + +++ +++ E76A +++ ++ +++ ++ ++ ++ ++ +E76K +++ +++ + +++ ++ + +++ +++ ++ nsp + K143E + ++ K206R ++ + ++ +++ + + R275G ++ +++ F278L ++ + + + D344N ++ ++ ++ + L351F ++ + +++ ++E397K + ++ + N415D ++ ++ + + A439T +++ + +++ ++ ++ ++ + E734G +++ + ++++++ ++ + E734K +++ ++ + ++ ++ ++  ++. ++ + nsp F749L + F749V ++ ++ +++ + + L30P ++ ++ + L30R +++ ++ + +++ ++ + +++ ++ + ++ ++ D452N +++ ++++ ++ + +++ +++ ++ ++ G504S +++ + + +++ ++ + ++ ++ +++ F285S ++ L311F++ + + A348V ++ +++ ++ nsp—non-specific amplification +—minor amount oftarget PCR fragment ++—normal amount of target PCR fragment +++—abundantamount of target PCR fragment

TABLE 5 The values of dissociation constant (Kd) determined foroligoduplex substrate and mutant Taq DNA polymerases in Example 2. EMSAmeasurements were performed at fixed 0.1 nM concentration of oligoduplexand 0.25-100 nM concentration gradient of polymerase. The WT-Taqpolymerase Kd is 1.71-3.97 nM. Mutant name Mutant (1^(st) name (2^(nd)screening) Frequency Kd, nM screening) Frequency Kd, nM E230K 33 0.37*F73V 5 4.56-4.92 L30P 32 3.08 K206R 5  2.42-12.72 L30R 2.43 D452N 181.14 L30P 3 3.08 G504S 15 6.03 H75R 3 0.57 L311F 5 3.20 E90K 3 1.53*E507A 5 1.14* K143E 3 4.09 E189K 4 0.40* L351F 3 0.85 F285S 3 2.63 E397K3 2.64 A348V 3 0.62 A439T 3 3.06 *the Kd values of wt-Taq, E230K, E507A,E189K, E90K are taken from Example 1 (Table 2).

TABLE 6 The summarized data on different PCR inhibition experimentsperformed with blood, SDS, GuHCL and heparin. The thresholdconcentrations of inhibitor under which specific 1.825 kbp DNA fragmentis still synthesized are indicated. Blood, GuHCl, heparin, UPS perEnzyme % (v/v) SDS, % (w/v) mM 25 μl of PCR Taq DNA pol. 0 0.0025 200.0025 Fermentas #EP0404 wt His-Tag 0 0.0025 0 0.001 E189K 8 0.0075 700.0025 E230K 4 0.005 70 0.0025 E507K 8 0.0075 70 0.0025 H28R 4 0.0025 400.0025 E90K 2 0.005 40 0.0025 E76K 2 0.005 40 0.0025 H676R 2 0.005 400.0025 L30R 2 0.005 40 0.0025 D452N 1 0.0075 20 0.00625 E734K 1 0.005 200.0025 D732G 1 0.005 20 0.0025 D551N 1 0.005 20 0.00625 I553V 1 0.007520 0.0025 G504S 1 0.005 20 0.00625 H75R 0.5 0.005 20 0.0025 E76G 0 0.00520 0.0025 E76A 0 0.005 20 0.0025 A348V 0 0.0025 0 0.001 A439T 0 0.005 200.0025 E734G 0 0.005 20 0

TABLE 7 Summary of Taq DNA polymerase mutants Resistance IncreasedIncreased to SYBR Resistance Resistance Resistance Resistance Mutationspeed affinity Green to blood to SDS to GuHCl to heparin H28R + + + +L30R + + + + G38R + F73V + H75R + + + E76A + + E76G + + + E76K + + + +E90K + + + + + E189K + + + + + + K206R + E230K + + + + + + E315K + +A348V + L351F + A439T + + D452N + + + + G504S + + + + E507A +E507K + + + + + + D551N + + + + L552R + + I553V + + + D578N + +H676R + + + + + Q680R + D732G + + + + E734G + E734K + + + F749V + H28R +E507K + + H28R + Q680R + + E507K + Q680R + + L552R + Q680R + + E230K +E507K + + E189K + E507K + + E315K + E507K + + E230K + E315K + + E507K +L552R + + E189K + E230K + + + E507K Summarized information from Tables1, 2, 4, 5, 6 and FIG. 19.

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1. A DNA polymerase mutant comprising a Taq DNA polymerase amino acidsequence with a mutation at one or more of the following selected aminoacid positions: E189K, E230K, E507K, H28R, L30R, G38R, F73V, H75R, E76A,E76G, E76K, E90K, K206R, E315K, A348V, L351F, A439T, D452N, G504S,E507A, D551N, L552R, I553V, D578N, H676R, Q680R, D732G, E734G, E734K,F749V; wherein the polymerase mutant exhibits relative to wild-type DNApolymerase increased polymerase speed, increased affinity to DNAsubstrate and/or increased resistance to a DNA polymerase inhibitor; andwherein, when the mutation is E507K in combination with two or morefurther mutations or the mutation is Q680R in combination with four ormore further mutations, at least one of the further mutations is at oneof the selected amino acid positions; and when the mutation is I553V,this is not in combination with D551S.
 2. The DNA polymerase mutantaccording to claim 1, wherein the amino acid sequence has a mutation atno more than three of the selected amino acid positions.
 3. The DNApolymerase mutant according to claim 2, wherein the amino acid sequencehas a mutation at only one of the selected amino acid positions.
 4. TheDNA polymerase mutant according to claim 2, wherein the amino acidsequence has a mutation at only two of the selected amino acidpositions.
 5. The DNA polymerase mutant according to claim 1, whichexhibits increased polymerase speed relative to wild-type DNApolymerase.
 6. The DNA polymerase mutant according to claim 5, whereinthe selected amino acid positions are E189K, E230K, E507K, H28R, L30R,F73V, H75R, E76A, E76G, E76K, E90K, K206R, A439T, D452N, G504S, D551N,I553V, H676R, D732G, E734G, F749V.
 7. The DNA polymerase mutantaccording to claim 5, which exhibits an increased polymerase speed whichis at least 1.5 times faster than wild-type DNA polymerase.
 8. The DNApolymerase mutant according to claim 1, which exhibits increasedaffinity to DNA substrate relative to wild-type DNA polymerase.
 9. TheDNA polymerase mutant according to claim 8, wherein the selected aminoacid positions are E189K, E230K, E507K, H75R, E315K, A348V, L351F,L552R, D578N.
 10. The DNA polymerase mutant according to claim 1, whichexhibits increased resistance to a DNA polymerase inhibitor selectedfrom the group consisting of SYBR Green I dye, blood, SDS, guanidiniumsalts and heparin.
 11. The DNA polymerase mutant according to claim 10,wherein the selected amino acid positions are E189K, E230K, E507K, H28R,L30R, G38R, H75R, E76A, E76G, E76K, E90K, E315K, A439T, D452N, G504S,E507A, D551N, L552R, I553V, D578N, H676R, Q680R, D732G, E734G, E734K.12. The DNA polymerase mutant according to claim 4, wherein the selectedamino acid positions are: H28R+E507K, H28R+Q680R, E507K+Q680R,L552R+Q680R, E230K+E507K, E189K+E507K, E315K+E507K, E230K+E315K,E507K+L552R.
 13. The DNA polymerase mutant according to claim 8, whereinthe Kd for a DNA oligoduplex substrate is no more than 1 nM, as measuredby electrophoretic shift mobility assay following incubation in 40 mMTris, 20 mM acetic acid, 1 mM EDTA at pH8.4, in the presence of 10% v/vglycerol at 4° C. for 30 mins.
 14. DNA polymerase mutant according toclaim 10, wherein the Kd for a DNA oligoduplex substrate in the presenceof 0.4 μM SYBR Green I dye is no more than 10 nM, as measured byelectrophoretic shift mobility assay following incubation in 40 mM Tris,20 mM acetic acid, 1 mM EDTA at pH8.4, in the presence of 10% v/vglycerol at 4° C. for 30 mins.
 15. The DNA polymerase mutant accordingto claim 1, wherein the selected amino acid positions are E189K, E230Kand E507K.
 16. A kit for nucleic acid amplification, which comprises aDNA polymerase mutant according to claim 1 and one or more reagents fora DNA synthesis reaction.
 17. Use of a DNA polymerase mutant accordingto claim 1, in a polymerase chain reaction.
 18. A process for theproduction of a DNA polymerase mutant according to claim 1, whichprocess comprises: (1) subjecting a polynucleotide encoding a DNApolymerase to error-prone PCR to generate a mutant library comprising anarray of differently-mutated polynucleotides; (2) screening the mutantlibrary for increased polymerase speed, increased polymerase affinity toDNA substrate or increase resistance to a DNA polymerase inhibitor; (3)selecting one or more mutant DNA polymerases from screening step 2; and(4) repeating steps 1 to 3 until a final DNA polymerase mutant isobtained.
 19. A DNA polymerase mutant obtained by the process of claim18.
 20. The DNA polymerase mutant according to claim 5, which exhibitsan increased polymerase speed which is at least 3 times faster thanwild-type DNA polymerase.
 21. The DNA polymerase mutant according toclaim 5, which exhibits an increased polymerase speed which is at least12 times faster than wild-type DNA polymerase.